The surface loops of proteins and bioactive peptides have often been implicated in recognition by protein binding partners. Accordingly, it is of interest to investigate these loops as potential leads for drug discovery. Particularly of interest are β-hairpins: The β-hairpin motif is very abundant in nature and occurs on the surface of many protein ligands and in the hypervariable domains of antibodies. The β-hairpin motif consists of two antiparallel β-strands linked by a short loop or turn and have been classified depending on the H-bonding network [Sibanda, B. L.; Blundell, T. L.; Thornton, J. M. J. Mol. Biol. 1989, 206, 759-777].
The ability to generate β-hairpin peptidomimetics using combinatorial and parallel synthesis methods has now been established (L. Jiang, K. Moehle, B. Dhanapal, D. Obrecht, J. A. Robinson, Helv. Chim. Acta. 2000, 83, 3097-3112). However these molecules may not be synthesized in libraries as large as 1010 or 1012.
A complementary strategy for peptide-based lead discovery consists of display of libraries on filamentous bacteriophage which allows the preparation of libraries as large as 1010-1012, many magnitudes larger than libraries that may be prepared synthetically.
Furthermore rapid and inexpensive selection protocols are available for identifying those library members that bind to a target of interest. Phage display technique allows the construction of cyclic constrained peptides such as disulfide-constrained β-hairpin loops, as is well known (H. B. Lowman, Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 401-24).
These loops represent a limited number of conformations which may result in isolation of affinity ligands for a receptor target. Cyclic peptides, however, stabilized by only one disulfide bond are still conformationally quite flexible. Also, it is well known that disulfide bond formation and cleavage can be reversible and flexibility is increased by the fact that the peptide constraints are fused to the amino terminus of the gene III protein. Thus it is important to stabilize such loop constructs by additional residues, adjacent to the disulfide bond which favor the β-sheet conformation. (R. H. Hoess, Current opinion in Structural Biology 1993, 3, 572-579). This may not lead to high affinity ligands for a receptor target. The same peptide loop is fixed in the natural protein scaffold by the protein scaffold on to N- and C-terminus of the loop and is additionally constrained by hydrogen bonds of anti-parallel beta-sheets which is induced by the natural protein scaffold. Other approaches have been proposed such as peptide scaffolds for turn display (A. G. Cochran, R. T. Tong, M. A. Starvasnik, E. J. Park, R. S. McDowell, J. E. Theaker, N. J. Skeleton, J. Am. Chem. Soc. 2001, 123, 625-632). Another possible solution to this problem is to use structural constraints of a folded protein to present small variable peptide segments (P.-A. Nygren, M. Uhlen, Curr. Opin. Struc. Biol. 1997, 7, 463-469; G. P. Smith, S. U. Patel, J. D. Windass, J. M. Thornton, G. Winter, A. D. Griffiths, J. Mol. Biol. 1998, 277, 317-332; A. Christmannn, K. Walter, A. Wenzel, R. Krazner, R. Kolmar, Protein Eng. 1999, 12, 797-806).
In fact the epitope transfer from proteins into small peptides remains a problem (A. G. Cochran Chem. Biol. 2000, 7, R85-R94).